There is a great need for the development of efficient and accurate systems for the diagnosis of a variety of medical conditions, disorders, and diseases. A means for rapid and accurate analysis and diagnosis of ex vivo bodily fluid samples at the point of care is particularly desirable. This requires an effective means for identifying in a patient the presence of specific chemical and/or biological agents including, but not limited to, nucleic acids, proteins, illicit drugs, toxins, pharmaceuticals, carcinogens, poisons, allergens, and infectious agents.
Current methods of detecting such chemical or biological agents entail extraction of a sample into organic solvents, followed by analysis using stand-alone analytical systems such as gas-liquid chromatography and/or mass spectroscopy. These methods are time-consuming and often expensive. Moreover, certain analytes/biomarkers of interest (i.e., nucleic acids, carcinogens, or toxins) are not readily detected by standard chemical tests utilized in a typical clinical physician's office or even in hospital departments. Further, on-site test devices for accurate analyte/biomarker detection are not presently available. The development of a biosensor device that could accurately and efficiently detect/screen for chemical and biological agents in bodily fluid samples would therefore provide a significant cost and time benefit.
Three recent advancements in medicine are particularly germane to expanding the potential of detecting chemical and/or biological agents, especially with regard to the diagnosis of disease: nanotechnology, biodetectors (biosensors), and the identification of biomarkers for specific diseases and/or conditions. Nanotechnology, such as nanoparticles, offers many advantages when used for applications such as sensor technology for detecting chemical agents. For example, nanoparticle-bound surfaces can form a sensor effective in detecting a target chemical agent.
The term “biodetectors” or “biosensors” relates to the use of naturally occurring and/or synthetic compounds as highly specific and extraordinarily sensitive detectors of various types of molecules and markers of disease. Naturally-occurring compounds such as antibodies have been used to provide molecular recognition for a wide variety of target molecules in diagnostic assays. Alternatively, synthetic compounds have been manufactured that mimic naturally-occurring mechanisms of DNA, RNA, and protein synthesis in cells to facilitate the detection of target chemical or biological agents.
Aptamers have recently been identified as potentially effective biosensors for molecules and compounds of scientific and commercial interest (see Brody, E. N. and L. Gold, “Aptamers as therapeutic and diagnostic agents,” J. Biotechnol., 74(1):5–13 (2000) and Brody et al., “The use of aptamers in large arrays for molecular diagnostics,” Mol. Diagn., 4(4):381–8 (1999)). For example, aptamers have demonstrated greater specificity and robustness than antibody-based diagnostic technologies. In contrast to antibodies, whose identification and production completely rest on animals and/or cultured cells, both the identification and production of aptamers takes place in vitro without any requirement for animals or cells.
Aptamer synthesis is potentially far cheaper and reproducible than antibody-based diagnostic tests. Aptamers are produced by solid phase chemical synthesis, an accurate and reproducible process with consistency among production batches. An aptamer can be produced in large quantities by polymerase chain reaction (PCR) and once the sequence is known, can be assembled from individual naturally occurring nucleotides and/or synthetic nucleotides. Aptamers are stable to long-term storage at room temperature, and, if denatured, aptamers can easily be renatured, a feature not shared by antibodies. Furthermore, aptamers have the potential to measure concentrations of ligand in orders of magnitude lower (parts per trillion or even quadrillion) than those antibody-based diagnostic tests. These inherent characteristics of aptamers make them attractive for diagnostic applications.
A number of “molecular beacons” (often fluorescence compounds) can be attached to aptamers to provide a means for signaling the presence of and quantifying a target chemical or biological agent. For instance, an aptamer specific for cocaine has recently been synthesized (Stojanovic; M. N. et al., “Aptamer-based folding fluorescent sensor for cocaine,” J. Am. Chem. Soc., 123(21):4928:31 (2001)). A fluorescence beacon, which quenches when cocaine is reversibly bound to the aptamer is used with a photodetector to quantify the concentration of cocaine present. Aptamer-based biosensors can be used repeatedly, in contrast to antibody-based tests that can be used only once.
Of particular interest as a beacon are amplifying fluorescent polymers (AFP). AFPs with a high specificity to TNT and DNT have been developed. It has been noted that a detector based on AFP technology, with high specificity to TNT and DNT, can also detect propofol, an intravenous anesthetic agent, in extremely low concentrations. The combination of AFP and aptamer technologies holds the promise of robust, reusable biosensors that can detect compounds in minute concentrations with high specificity.
The term “biomarker” refers to a biochemical in the body that has a particular molecular trait to make it useful for diagnosing a condition, disorder, or disease and for measuring or indicating the effects or progress of a condition, disorder, or disease. For example, common biomarkers found in a person's bodily fluids (i.e., breath or blood), and the respective diagnostic conditions of the person providing such biomarkers include, but are not limited to, acetaldehyde (source: ethanol; diagnosis: intoxication), acetone (source: acetoacetate; diagnosis: diet; ketogenic/diabetes), ammonia (source: deamination of amino acids; diagnosis: uremia and liver disease), CO (carbon monoxide) (source: CH2Cl2, elevated % COH; diagnosis: indoor air pollution), chloroform (source: halogenated compound), dichlorobenzene (source: halogenated compounds), diethylamine (source: choline; diagnosis: intestinal bacterial overgrowth), H (hydrogen) (source: intestines; diagnosis: lactose intolerance), isoprene (source: fatty acid; diagnosis: metabolic stress), methanethiol (source: methionine; diagnosis: intestinal bacterial overgrowth), methylethylketone (source: fatty acid; diagnosis: indoor air pollution/diet), O-toluidine (source: carcinoma, metabolite; diagnosis: bronchogenic carcinoma), pentane sulfides and sulfides (source: lipid peroxidation; diagnosis: myocardial infarction), H2S (source: metabolism; diagnosis: periodontal disease/ovulation), MeS (sucrose: metabolism; diagnosis: cirrhosis), and Me2S (source: infection; diagnosis: trench mouth).
Mechanisms of drug metabolism are extremely complex and are influenced by a number of factors including competitive binding on protein and red blood cells with other molecules, enzymatic activity, particularly in the liver, protein, and red blood cell concentration and a myriad of other factors. Currently, very little technology is available that can measure drug concentration in a patient in real-time, especially at the point of care, and thereby allow convenient determination of pharmacokinetics and pharmacodynamics of multiple compounds in real-time.
Accordingly, there are a number of medical conditions that can be monitored by detecting and/or measuring biomarkers present in a person's breath (including breath condensates or aerosolized particles) or other bodily fluids. While there has been technology generated towards the synthesis and use of aptamers and other multimolecular devices such as biosensors, very little technology exists to address the use of aptamers, or other biodetectors, in combination with nanoparticles to form sensors for the ex vivo diagnosis of disease and/or detection of a naturally occurring or synthetic compounds. It is therefore desirable to provide a low-cost means for accurately and timely detecting and/or measuring the presence of metabolites in a person's bodily fluids in low concentrations.